A number of immunotherapeutic agents have been described for use in cancer treatment, including therapeutic monoclonal antibodies (mAbs), immunoconjugated mAbs, radioconjugated mAbs and bi-specific T-cell engagers.
Typically these immunotherapeutic agents target a single antigen: for instance, Rituximab targets CD20; Myelotarg targets CD33; and Alemtuzumab targets CD52.
However, it is relatively rare for the presence (or absence) of a single antigen effectively to describe a cancer, which can lead to a lack of specificity.
Most cancers cannot be differentiated from normal tissues on the basis of a single antigen. Hence, considerable “on-target off-tumour” toxicity occurs whereby normal tissues are damaged by the therapy. For instance, whilst targeting CD20 to treat B-cell lymphomas with Rituximab, the entire normal B-cell compartment is depleted, whilst targeting CD52 to treat chronic lymphocytic leukaemia, the entire lymphoid compartment is depleted, whilst targeting CD33 to treat acute myeloid leukaemia, the entire myeloid compartment is damaged etc. The predicted problem of “on-target off-tumour” toxicity has been borne out by clinical trials. For example, an approach targeting ERBB2 caused death to a patient with colon cancer metastatic to the lungs and liver. ERBB2 is over-expressed in colon cancer in some patients, but it is also expressed on several normal tissues, including heart and normal vasculature.
For some cancers, targeting the presence of two cancer antigens may be more selective and therefore effective than targeting one. For example, B-chronic lymphocytic leukaemia (B-CLL) is a common leukaemia which is currently treated by targeting CD19. This treats the lymphoma but also depletes the entire B-cell compartment such that the treatment has a considerable toxic effect. B-CLL has an unusual phenotype in that CD5 and CD19 are co-expressed. By targeting only cells which express CD5 and CD19, it would be possible to considerably reduce on-target off-tumour toxicity.
There is thus a need for immunotherapeutic agents which are capable of more targeting to reflect the complex pattern of marker expression that is associated with many cancers.
Chimeric Antigen Receptors (CARs)
Chimeric antigen receptors are proteins which graft the specificity of a monoclonal antibody (mAb) to the effector function of a T-cell. Their usual form is that of a type I transmembrane domain protein with an antigen recognizing amino terminus, a spacer, a transmembrane domain all connected to a compound endodomain which transmits T-cell survival and activation signals (see FIG. 1A).
The most common form of these molecules are fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies which recognize a target antigen, fused via a spacer and a trans-membrane domain to a signaling endodomain. Such molecules result in activation of the T-cell in response to recognition by the scFv of its target. When T cells express such a CAR, they recognize and kill target cells that express the target antigen. Several CARs have been developed against tumour associated antigens, and adoptive transfer approaches using such CAR-expressing T cells are currently in clinical trial for the treatment of various cancers.
However, the use of CAR-expressing T cells is also associated with on-target, off tumour toxicity. For example, a CAR-based approach targeting carboxy anyhydrase-IX (CAIX) to treat renal cell carcinoma resulted in liver toxicity which is thought to be caused by the specific attack on bile duct epithelial cells (Lamers et al (2013) Mol. Ther. 21:904-912).
Dual Targeting CAR Approaches
In order to address the problem of “on target, off tumour” toxicity, CAR T cells have been developed with dual antigen specificity. In the “dual targeting” approach, two complementary CARs are co-expressed in the same T-cell population, each directed to a distant tumour target and engineered to provide complementary signals.
Wlikie et al (2012 J Clin Immunol 32:1059-1070) describe a dual targeting approach in which ErbB2- and MUC1-specific CARs are co-expressed. The ErbB2-specific CAR provided the CD3ζ signal only and the MUC1-specific CAR provided the CD28 co-stimulatory signal only. It was found that complementary signalling occurred in the presence of both antigens, leading to IL-2 production. However, IL-2 production was modest when compared to control CAR-engineered T cells in which signaling is delivered by a fused CD28+CD3ζ endodomain.
A similar approach was described by Kloss et at (2013 Nature Biotechnol. 31:71-75) in which a CD-19 specific CAR was used which provides a CD3ζ-mediated activation signal in combination with a chimeric co-stimulatory receptor specific for PSMA. With this ‘co-CAR’ design, the CAR T-cell receives an activation signal when it encounters a target cell with one antigen, and a co-stimulatory signal when it encounters a target cell with the other antigen, and only receives both activatory and co-stimulatory signals upon encountering target cells bearing both antigens.
This represents an early attempt at restricting CAR activity to only a target cell bearing two antigens. This approach however is limited: although CAR T-cell activity will be greatest against targets expressing both antigens, CAR T-cells will still kill targets expressing only antigen recognized by the activatory CAR; further, co-stimulation results in prolonged effects on T-cells which last long after release of target cell. Hence, activity against single-antigen positive T-cells equal to that against double-positives might be possible for example in a situation where single-positive tissues are adjacent to, or in a migratory path from double positive tumour.
There is thus a need for improved CAR-based therapeutic approaches with reduced on-target off-tumour toxicity where T-cell activation is wholly restricted to target cells which express both antigens.